The invention concerns protein glycosylation, and in particular, the glycosylation of protein or polypeptide substrates using human or animal GalNAc-T4 transferase. In another aspect, the invention concerns the generation and high-level secreted expression of glycosyltransferase enzymes, and other transmembrane proteins, in mammalian host cells.
Glycosylation is the addition of glycan moieties to proteins. The initial steps of glycosylation involve recognition events between protein and a glycosyltransferase, which events determine the sites of glycan attachment. Different glycosyltransferases have been isolated and identified, and a number of specific sites of glycan addition to proteins have been determined.
The glycosylation of serine and threonine residues during mucin-type O-linked protein glycosylation is catalyzed by a family of polypeptide GalNAc transferases (EC 2.4.1.41). Five distinct GalNAc-transferase genes termed GalNAc-T1, -T2, -T3, -T4 and -T5 have been cloned and characterized. Homa et al., J. Biol. Chem. 268:12609 (1993); Hagen et al., J. Biol. Chem. 273:8268 (1998); White et al., J. Biol. Chem.270: 24156 (1995); Bennett et al., J. Biol. Chem. 271:17006 (1996); Bennett et al., Glycobiology 8:547 (1998); Hagen et al., J. Biol. Chem. 272:13843 (1997); Hagen et al., J. Biol. Chem. 273:27749 (1998). The GalNAc transferases characterized to date have distinct acceptor substrate specificities. Bennett et al. (1996), supra; Wandall et al., J. Biol. Chem. 272:23503 (1997); Bennett et al. (1998), supra. Recent findings have suggested that the GalNAc-transferases comprise a gene family and that each GalNAc transferase has distinct functions.
Isolation of GalNAc-T3 and use of the GalNAc-T3 polypeptide to glycosylate substrates are disclosed in copending application Ser. No. 648,298, filed May 15, 1996, the disclosure of which is incorporated by reference.
A putative murine orthologue of GalNAc-T4 is described in Hagen et al., J. Biol. Chem. 272:13843 (1997). The murine GalNAc-T4 was tested with a small panel of peptides, including two sequences from the tandem repeat of MUC1, and no activity with those substrates was found in that study. O-glycosylation of the cancer-associated cell membrane mucin, MUC1, has attracted attention because it is altered in cancer cells with smaller and fewer glycans. Taylor-Papadimitriou et al., Ann. N.Y. Acad. Sci. 690:69 (1993); Lloyd et al., J. Biol. Chem. 271:33325 (1996); Brockhausen, Eur. J. Biochem. 233:607 (1995). The change in O-glycosylation leads to exposure of cancer-associated epitopes within the tandem repeat region of MUC1. Analysis of the in vitro O-glycosylation properties of various GalNAc-transferase preparations, including purified GalNAc-T1, GalNAc-T2 and GalNAc-T3 suggests that only three of five possible sites in the repeat are glycosylated. Wandall et al. (1997), supra.
The cell adhesion molecule, P-selection, binds to its ligand, the P-selection glycoprotein ligand 1 (PSGL-1) through interaction with an O-glycan at threonine 57. Liu et al., J. Biol. Chem. 273:7078 (1998). Previous unpublished findings have suggested that GalNAc-T1, GalNAc-T2 and GalNAc-T3 do not utilize this substrate.
The use of GalNAc-T4 is disclosed for the O-glycosylation of substrates. It has been found that GalNAc-T4 exhibits a different substrate specificity than previously-characterized GalNAc-transferases.
The unique specificity of human GalNAc-T4 is exemplified by its ability to glycosylate specific serine and threonine residues in MUC1 tandem repeat and PSGL-1. For example, GalNAc-T4 glycosylates two sites in the MUC1 tandem repeat sequence using MUC1 tandem derived glycopeptides. Further characterizing the unique activity of GalNAc-T4 is its ability to glycosylate synthetic peptides derived from PSGL-1.
In a preferred embodiment, a method of glycosylation is disclosed which comprises glycosylation of substrates with GalNAc-T4, used in combination with GalNAc-T1, GalNAc-T2 and/or GalNAc-T3.
Substrates which are glycosylated in accordance with the method of the present invention are useful for the preparation of glycoprotein-based vaccines and anti-inflammatory agents.
In a related method, soluble GalNAc-T4, or soluble forms of other type II transmembrane proteins, are produced and detected in animal cells with high-level expression. In this embodiment, a truncated form of the protein, which excludes the N-terminal hydrophobic signal sequence, is provided in association with a suitable secretion signal, SEQ ID NO:10. Host cells expressing the soluble form of protein are detected by screening with an antibody raised against the soluble form of the protein.